Bacterial and viral superantigens (SAGS) represent some of the most potent toxins known to exist and can cause massive overstimulation of the host immune system, initially through cytokine release, T cell proliferation, and finally through T cell anergy and apoptosis (Marrack and Kappler, 1990, Science 248:1066; Ulrich et al., 1995, Trends Microbiol. 3: 463-468; Papageorgiou and Acharya, 2000, Trends Micobiol 8: 369-375).
Superantigens secreted by the bacterial pathogen Staphyloccocus aureus (S. aureus) overstimulate the host immune system by binding as intact proteins to the DRα1 domain of the MHC class II receptor and the TCRVβ domain from the T cell receptor, thereby activating a large population of T cells and causing the excessive release of a number of cytokines. Initially, S. aureus SAGs compromise the immune system by inducing the large-scale release of cytokines, such as IL-2, TNF-α, and IFN-γ, and the hyperproliferation of T cells. These events are eventually followed by the deletion of the affected T cell population through apoptosis (see FIG. 1A).
Superantigens are the causative agents in the acute diseases, food poisoning and toxic shock syndrome, and in more chronic conditions, such as inflammatory skin diseases. In addition to the toll on public health, S. aureus superantigens also represent a potential biothreat to national security (Schiffenbauer et al., 1993, Proc. Natl. Acad. Sci. USA 90: 8543-8546; Laurence et al., 1992, Nature 358: 255-9; Yarwood et al., 2000, FEMS Microbiol. Lett. 192: 1-7).
Human diseases resulting from Staphylococcal and streptococcal infection and the resultant release of SAGs into the infected individual are primarily characterized by fever and shock and continue to present a major health problem worldwide. The S. aureus enterotoxins (SEA-I) are thought to be the causative agents in 33% of all food poisoning cases (Chesney et al., 1984, A. Rev. Microbiol. 38: 315-338) and are the most frequent cause of hospital-acquired infections (Emori and Gaynes, 1993, Clin. Microbiol. Rev. 6: 428-442). An estimated 1.3 million US patients acquire S. aureus infections annually, generally resulting in doubling the length of hospitalization and the associated medical costs.
Toxic Shock Syndrome (TSS), mediated primarily by the S. aureus TSST-1 toxin, was recognized as a significant problem in the 1980s when more than a thousand cases of TSS became linked to tampon usage (Davis et al., 1980, New Engl. J. Med. 303: 1429-1435). Presently, potentially life-threatening TSS most frequently occurs when S. aureus bacteria infect surgical wounds or injury sites, with up to 6000 cases each year in the US (Schlievert et al., 1988, European Conference on Toxic Shock Syndrome. Intl. Congress and Symposium Series, Vol. 229. Royal Society of Medicine Press, New York; Bohach et al., 1990, Crit. Rev. Microbiol. 17: 251-272). In more chronic conditions, such as inflammatory skin diseases, a growing body of evidence implicates S. aureus SAGs in the onset of these diseases by disrupting immune activity through abnormal T cell activation and inflammatory cytokine release.
Some studies have speculated that SAGs promote autoimmune and immunodeficiency diseases such as multiple sclerosis (Schiffenbauer et al., 1993, supra) and HIV infection (Laurence et al., 1992, supra) by continually weakening the normal host immune response, thus allowing the onset of disease to progress more quickly.
The mechanism of S. aureus pathogenesis has been attributed to an alternate mode of SAG binding. In contrast to foreign antigens, SAGs bypass the internalization and processing by the antigen presenting cell (APC), and instead bind externally to the DRα1 domain of the MHC class II receptor on APCs and the Vβ domain of the T cell receptor (TCR) on T cells (Jardetzky, et al., 1994, Nature 368: 711-8; Kim et al., 1994, Science 266: 1870-4; Fields et al., 1996, Nature 384: 188-92; Li et al., 1998, Immunity 9: 807-816). The alternate binding of SAG to the MHC class II-TCR complex is followed by two additional signaling events between APCs and T cells, the engagement of co-stimulatory ligands and their cognate receptors, such as the B7 ligand and CD28 (Reiser et al., 1996, N Engl J Med 335: 1369-77), and the involvement of the autocrine and paracrine cytokine network (see FIG. 1A) (Haddad, J., 2002, Biochem. Biophys. Res. Comm. 297: 700-13). SAGs also have the ability to bind to multiple isoforms of TCRVβ. (Choi et al., 1989, Proc Natl Acad Sci U S A 86: 8941-5) and can activate up to 20% of T cells compared to 0.0001% by a conventional antigen, resulting in the activation of a large population of T cells (Sundberg and Mariuzza, 2002, Curr. Opin. Immunol. 14: 36-44). Crystal structures of SAGs (Papageorgiou et al., 1998, J. Mol. Biol. 277: 61-79; Prasad et al., 1997, Protein Sci. 6: 1220-7; Chi et al., 2002) J. Biol. Chem. 277: 22839-46) and their complexes with the MHC class II and T cell receptors have been reported.
SAGs simultaneously bind to the outside surfaces of the HLA-DRα domain of MHC class II and the Vβ domain of the T cell receptors, primarily through contacts with the complementarity-determining regions (CDR) 1 and 2 and the hypervariable region HV4 (Choi et al., 1989, Proc. Natl. Acad. Sci. USA 86: 8941-8945; Kappler et al., 1989, Science 244: 811-813; Fraser, 1989, Nature 339: 221-223). X-ray crystallographic studies have provided a very detailed structural understanding of how the SAGs contact the MHC class II and T cell receptors, leading to a better understanding of the mechanism of SAG stimulation (Jardetzky et al., 1994, Nature 368: 711-718; Kim et al., 1994, Science 266: 1870-1874; Fields et al, 1996, Nature 384: 188-192).
Generally stimulation is achieved by SAG binding to multiple TCRVβ isoforms and activation of larger numbers of T cells than occur during foreign-peptide stimulation. This over-stimulation of the immune system results in massive cytokine release, including IL-2 and IFN-γ from the T cells, and IL-1β and TNF-α from APCs (Langford et al., 1978, Infect. Immun. 22: 62-68; Marrack et al., 1990, J. Exp. Med. 171: 455-464; Miethke et al., 1992, J. Exp. Med. 175: 91-98). The stimulated T cell population initially undergoes cell proliferation; however, in the course of 1-2 weeks, the affected T cells become anergic and are no longer able to respond to foreign and harmful agents. These T cells are eventually deleted from the immune cell repertoire through apoptosis or programmed cell death (Ettinger et al., 1995, J. Immunol. 154: 4302-4308).
Presently, ten S. aureus SAGs, SEA,B,C,D,E,F,G,H,I and TSST-1 and seven SAGs in Streptococcus pyogenes, SPE-A, SPE-C, SPE-G, SPE-H, SSA, SMEZ1, and SMEZ2 have been identified (Fraser, 2000, supra). Phylogenetic analysis of Staphylococcal and Streptococcal SAGs indicates a 20%-90% sequence similarity and suggests that all the toxins have evolved from a common ancestral gene. Very small amounts of purified toxin are capable of stimulating significant host effects. Small quantities of toxin (femtogram to picogram) can activate a culture of human peripheral blood lymphocytes and stimulate T cell proliferation of restricted TCRVβ domains (Carlsson and Sjogren, 1985, Cell Immunol. 96: 175-183). Oral injection of several micrograms of toxin is capable of inducing vomiting and diarrhea in humans and primates.
Treatment of SAG-based diseases is primarily limited to supportive therapy once symptoms have manifested. These treatments include antibiotic use, administration of intravenous fluids, and medications to treat low blood pressure and shock. Unfortunately, the heavy usage of antibiotics over the last several decades have led to the emergence of S. aureus strains resistant to the two most commonly-used antibiotics, methicillin (Kreiswirth et al., 1993, Science 259: 227-230) and vancomycin (Pearson, 2002, Nature 418: 469). Antibiotic resistance and the relative ineffectiveness of supportive treatments in cases of high load bacterial infections have heightened efforts to develop innovative strategies for combating Staphylococcal infections. In addition, given that SAGs are considered to be potential biowarfare select agents according to the CDC and can severely impair the ability of infected soldiers to perform in the battle field, there is a considerable and timely need to develop the next generation of countermeasures (i.e., vaccines and therapeutics) for both treatment of established disease and as a preventive measure against exposure to SAGs.
Toxic Shock Syndrome (TSS) is an acute, life-threatening condition caused by S. aureus infection, and is characterized by fever, hypotension, rash, multiorgan dysfunction, and desquamation during the early convalescent period. TSS may be caused by any of the superantigens produced by S. aureus, with TSST-1 and enterotoxin B being the first and second most frequently implicated.
Beyond supportive therapy, a number of different therapeutic strategies are being evaluated. Generally, these strategies are aimed at blocking the initial stages of pathogenesis prior to activation of T cell proliferation and cytokine release, and include neutralizing antibodies against S. aureus or SAGs, SAG-derived peptides that interfere with immune cell signaling and elicit anti-SAG-antibody production, transcriptional and translational inhibition of SAG genes, receptor-based countermeasures to inhibit SAG binding to immune cells, inhibition of co-stimulatory signals that are required for T cell stimulation, IL-10 cytokine-based approaches, small molecule inhibitors, and anti-cytokine antibodies. For example, one particular approach targets the downstream effects of SAG-induced cytokine release by using anti-cytokine antibodies to TNFα and IFNγ. However, such anti-cytokine strategies may prove ineffective or problematic in view of the massive amounts of different cytokines that are released and the multiple signaling pathways that become activated in response to the SAG induced cytokines (Miethke et al., 1992, supra; Matthys et al., 1995, Infect. Immun. 63:1 158-1164).
It has previously been reported that a DRα1-(GSTAPPA)2-TCRVβ3 chimera inhibited SEB-induced IL-2 cytokine release and T cell proliferation in a mixture of human peripheral mononuclear and dendritic cells (Lehnert et al., 2001, Biochemistry 40: 4222-4228). The object of the present invention is to provide effective therapeutic strategies against Staphylcoccal and Streptococcal SAGs.